AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H1906-H1916, 2007. First published December 8, 2006; doi:10.1152/ajpheart.00793.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/H1906    most recent
00793.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schroder, E.
Right arrow Articles by Satin, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schroder, E.
Right arrow Articles by Satin, J.

Chronic verapamil treatment remodels ICa,L in mouse ventricle

Elizabeth Schroder,1 Janos Magyar,1 Don Burgess,2 Douglas Andres,3 and Jonathan Satin1

1Department of Physiology, University of Kentucky, Lexington; 2Department of Physics, Asbury College, Wilmore; and 3Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky

Submitted 24 July 2006 ; accepted in final form 5 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study we tested the hypothesis that ventricular homeostasis of L-type Ca2+ current (ICa,L) minimally involves regulation of the main pore-forming {alpha}-subunit (CaV1.2) and auxiliary proteins that serve as positive or negative regulators of ICa,L. We treated animals for 24 h with verapamil (Ver, 3.6 mg·kg–1·day–1), isoproterenol (Iso, 30 mg·kg–1·day–1), or Iso + Ver via osmotic minipumps. To test for alterations of Ca2+ channel complex components we performed real-time PCR and Western blot analysis on ventricle. In addition, cardiac myocytes (CMs) were dispersed and current was recorded in the whole cell configuration to evaluate ICa,L. Surprisingly, 24- to 48-h Ver increased CaV1.2 mRNA and protein and ICa,L current (Ver 11 ± 1pA/pF vs. control 7 ± 0.5pA/pF; P < 0.01). ICa,L from CMs in Ver mice showed no change in whole cell capacitance. To examine the in vivo effects of a physiologically relevant Ca2+ channel agonist, we treated mice with Iso. Twenty-four-hour Iso infusion increased heart rate; CaV1.2- and CaVbeta2 mRNA levels were constant, but the Ca2+ channel subunit mRNA Rem was increased twofold. Cells isolated from 24-h Iso hearts showed no change in basal ICa,L density and diminished responsiveness to acute 1 µM Iso. To further examine the homeostatic regulation of the Ca2+ channel, we treated animals for 24 h with Iso + Ver. The influence of Iso + Ver was similar that of to Iso alone on Ca2+ channel mRNAs and ICa,L, with the exception that it prevented the increase in Rem seen with Iso treatment. Long-term Ca2+ channel blockade induces an increase of CaV1.2 mRNA and protein and significantly increases ICa,L.

calcium channel blockade; ventricle; electrocardiogram; electrophysiology

THE L-TYPE CA2+ CHANNEL CaV1.2, the predominant voltage-gated Ca2+ channel in mammalian myocardium, provides the Ca2+ flux necessary to initiate excitation-contraction coupling. A variety of conditions chronically regulate excitability via alterations of ion channel density. For example, changes in transient outward current levels across the ventricular wall can lead to arrhythmias (1). CaV1.2 current (ICa,L) studies show less consistent results in a variety of disease models culminating in cellular hypertrophy (41), although in late-stage heart failure the consensus finding is that ICa,L declines (47). The unchanged level of ICa,L in all but late-stage heart failure suggests that ventricular myocytes may have the capacity to homeostatically regulate ICa,L.

At the plasma membrane the Ca2+ channel complex exists as a heteromultimer consisting of the {alpha}1-subunit (CaV1.2), a cytosolic beta-subunit (CaVbeta2), and a covalently linked {alpha}2{delta}-subunit. CaV1.2, the pore-forming subunit, determines ion selectivity and voltage sensitivity and is a binding site for clinically used Ca2+ channel blockers. CaVbeta coexpression primarily increases CaV1.2 current density in heterologous expression systems (4, 8, 21, 48) and in myocytes that overexpress CaVbeta (45). In addition to increasing functional expression, CaVbetas alter channel kinetics (29, 36, 44) and voltage dependence (5, 28, 39). Among the accessory subunits, the CaVbetas are the only accessory proteins known to increase current density. In contrast to CaVbeta, the {alpha}2{delta}-subunit has modest effects on channel current density and has variable effects on channel voltage dependence and kinetics (6, 25, 26, 31). Overall, CaVbeta2 in the heart serves as a positive regulator of ICa,L (11). We recently showed (13, 19) that in heterologous expression systems small G proteins of the RGK class such as Rem bind to CaVbeta2 and inhibit ICa,L expression. Other members of the RGK family include Rad, Rem2, and Gem/Kir, which have recently been identified as novel contributors to the regulation of L-type Ca2+ channel function. Thus both positive and negative regulators, in the form of auxiliary proteins, influence ultimate ICa,L density.

Ca2+ channel blockers (CCBs) are frequently used to regulate blood pressure in hypertensive patients (22, 32, 38); however, little is known about the long-term cardiac effects of these drugs on cardiac myocytes. The primary objective of this study was to test the hypothesis that cardiac ICa,L is homeostatically regulated. We postulated that Ca2+ entering through the L-type channel provides feedback regulation. According to our hypothesis, decreasing ICa,L, such as via chronic CCB, would elicit upregulation of ICa,L. Conversely, stressors that acutely increase cytosolic Ca2+ cause long-term decreases of ICa,L as in heart failure (7, 46). Our results demonstrate that long-term Ca2+ channel blockade induces an increase of CaV1.2 mRNA and protein and significantly increases basal ICa,L. In contrast, chronic beta-adrenergic stimulation induces upregulation of positive and negative regulators of ICa,L with no significant change in basal ICa,L. Together, these results suggest that the main pore-forming subunit plays a greater role in determining ICa,L than auxiliary subunits.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mini-osmotic pumps. Female or male ICR mice (12–14 wk of age) weighing between 25 and 30 g were anesthetized with a ketamine-xylazine mixture (ip), allowing the subcutaneous implantation of mini-osmotic pumps (Alzet, model 2001). The pumps were filled with verapamil (Ver), isoproterenol (Iso), isoproterenol and verapamil (Iso + Ver), or vehicle alone (0.02% ascorbic acid). Mice carrying drug-loaded minipumps are abbreviated as Iso, Ver, and Iso + Ver for pumps containing isoproterenol, verapamil, or the two drugs together. Control animals carried minipumps with vehicle, and control animals were investigated in parallel with each set of experimental animals. Minipumps delivered Iso at 30 mg·kg–1·day–1, Ver at 3.6 mg·kg–1·day–1, or a combination of Iso and Ver for 24 h. After treatment animals were anesthetized and weighed. Hearts were excised, rinsed, blotted dry, weighed, and then frozen on dry ice and stored at –80°C until being studied. To account for sex-related responses a parallel study of male versus female mice resulted in similar changes in mRNA following mini-osmotic pump implantation and treatment for 24 h (data not shown). Animals were anesthetized and euthanized according to animal protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, revised 1996).

RNA extraction. Left ventricular free wall from male or female mice was rapidly excised and either snap frozen at –80°C or used immediately for RNA isolation. Total RNA was isolated with the RNAqueous-4PCR kit (Ambion) and quantitated spectrophotometrically at 260 nm. Contaminating genomic DNA was eliminated by DNase treatment (Ambion). A portion of the resulting RNA (1 µg) was immediately used as a template for cDNA synthesis. Reverse transcription was performed with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Removal of genomic DNA was confirmed by preparing a no-reverse transcription control for each sample. cDNA was then stored at –20°C.

Real-time PCR. Real-time PCR was performed in 96-well optical plates in triplicate with an ABI 7700 Sequence Detector. Samples (0.2 ng cDNA) were prepared with the TaqMan PCR core reagent kit (Applied Biosystems). Samples were cycled for 50 cycles with an ABI 7700 Sequence Detector (Applied Biosystems). Cycle conditions were as follows: 2 min at 50°C, 10 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. Primers were chosen that provided an efficiency of 90–100%. In all cases a single amplicon of the appropriate size was detected with gel electrophoresis. Results are expressed normalized to cyclophilin A according to the 2Formula ( where Ct is threshold cycle) method (20). No-reverse transcription controls were prepared for each sample and showed no amplification.

Western blots. Whole cell lysates were prepared from cells isolated from adult hearts treated for 48 h with Ver (n = 5) or vehicle (n = 4). SDS-PAGE (7.5% separating gel, Bio-Rad) and immunoblotting were carried out with routine protocols. Affinity-purified L-type Ca2+ channel {alpha}-subunit polyclonal antibody at 2 µg/ml antibody (D. Andres lab) was visualized with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Chemicon) and Super Signal West Pico Chemiluminescence (Pierce). Each lane contained 10 µg of total protein. All Western blot experiments were repeated at least three times to ensure that experimental observations were reproducible. Loading was confirmed by stripping (Restore Western Blot Stripping Buffer, Pierce) and reprobing blots with GAPDH monoclonal antibody (Ambion). Immunoblots were scanned on an Epson Perfection 1650 and quantified with densitometry (Scion Image, Scion).

Blood pressure and heart rate. Systolic and diastolic blood pressure (BP) and heart rate (HR) were measured in conscious, restrained mice with the Visitech Tailcuff System (Visitech Systems). BP parameters and heart weight-to-body weight ratios are shown in Table 1. No significant difference was observed between background and vehicle groups for the aforementioned physiological parameters. Therefore, in Figs. 1–7 and Table 2 these data were combined and designated as the control group.


View this table:
[in this window]
[in a new window]

  		Table 1. Physiological parameters in conscious ICR mice

 

Figure 1
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 1. Cardiac L-type and auxiliary subunit remodeling evaluated by real-time RT-PCR quantification in mice treated chronically with verapamil (Ver) for 1 day. Values are expressed as the relative quantification of the gene of interest vs. the internal control (cyclophilin A) and are represented as means ± SE. The numbers of mice used for experiments are shown within bars (*P < 0.05). Ct, threshold cycle.

 

Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 7. Representative averaged ECG in unrestrained mice showing T and P waves in both vehicle (A and C)- and Ver (B and D)-treated mice before (A and B) and after (C and D) acute-beta-stim. E: animal-by-animal comparison of the corrected QT (QTc) interval in vehicle- and Ver-treated animals.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Electrocardiogram parameters in conscious ICR mice

 
ECG. ECGs were measured noninvasively with an EC Genie (Mouse Specifics). Briefly, animals were placed on a gel electrode lead plate on the ECG recording platform. Animals were allowed to acclimate for 4–10 min. After the acclimation period, ECG signals were recorded for 2–10 min. Raw ECG data were signal averaged across successive R-R intervals. As necessary, a cotton swab was used to gently move the animal's feet into a proper position on the lead plate for recording. Baseline ECGs were recorded before Ver exposure and daily after exposure. ECGs were recorded 24 h after administration. Mice then received an intraperitoneal injection of Iso (30 mg/kg) followed by immediate ECG measurement. ECG parameters in conscious mice were taken from averaged ECG data. Because there is some question on the exact location of the T wave, especially T wave termination, we operationally defined a T wave as shown in Fig. 7: T is the first trough following the prominent QRS complex. Corrected QT interval (QTc) was calculated for mice following Mitchell et al. (35).

Cell isolation procedures. Single ventricular myocytes were enzymatically isolated with a modified Alliance for Cellular Signaling protocol (PP00000125) at 48 h after pump implantation. Myocytes were isolated 48 h after minipump implantation to allow time for any changes in transcription to be reflected at the functional protein level. Briefly, mice were anesthetized and hearts were rapidly excised and retrogradely perfused at 3 ml/min and 37°C for 4–8 min with a Ca2+-free bicarbonate-based perfusion buffer containing (mM) 113 NaCl, 4.7 KCl, 0.6 KH2PO4, 1.2 MgSO4, 0.6 NaH2PO4, 5.5 glucose, 12 NaHCO3, 10 KHCO3, 10 HEPES, 0.032 phenol red, 10 2,3-butanedione monoxime, and 30 taurine. The perfusion buffer was gassed with 95% O2-5% CO2 for at least 30 min before use. Enzymatic digestion began with 0.25 mg/ml liberase Blendzyme (Roche) and 12.5 µM CaCl2 added to the perfusion buffer for ~13 min until the heart was swollen and pale in color. The heart was then cut from the cannula. Ventricular tissue was placed in a dish with enzyme buffer and gently dissociated for several minutes. After the addition of stop buffer (perfusion buffer containing 10% FBS and 12.5 µM CaCl2), dissociation continued until large pieces of heart tissue were gently dispersed into the cell suspension. Cells were allowed to sediment by gravity for 10 min, followed by centrifugation at 180 rcf for 1 min. Cells were resuspended in perfusion buffer containing 5% FBS and 12.5 µM CaCl2. External Ca2+ was added incrementally back to the solution to 2.0 mM. Only rod-shaped, quiescent myocytes with clear edges were selected for current recording.

Ca2+ current measurements. Ca2+ current was recorded from Ca2+-tolerant female mouse ventricular cells at 37°C 1–6 h after isolation. After establishment of whole cell configuration, the cells were perfused with Na+- and K+-free solution containing (mM) 140 N-methyl-D-glucamine, 1.0 MgCl2, 2.5 CaCl2, 10 HEPES, 10 glucose, and 5 4-aminopyridine at pH 7.4. Pipettes had tip resistances of 1–2 M{Omega} after filling with pipette solution composed of (mM) 125 CsCl, 10 tetraethylammonium chloride Cl, 1.0 MgCl2, 10 EGTA, 5 Mg-ATP, and 5 HEPES (pH 7.2). Current was recorded with an Axopatch-200B amplifier (Axon Instruments). The series resistance was typically 4–8 M{Omega} before compensation (usually 50–75%). Outputs from the clamp amplifier were digitized at 20 kHz with an analog-to-digital converter (Digidata-1200, Axon Instruments) under software control (pCLAMP 8.2, Axon Instruments).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Persistent Ca2+ channel blockade increases CaV1.2 mRNA and protein and ICa,L. To examine homeostatic regulation of the Ca2+ channel in ventricular myocardium, we treated mice for 24 h with the L-type Ca2+ channel blocker Ver (3 mg·kg–1·day–1). Ver treatment resulted in an increase in CaV1.2 mRNA with no significant change in CaVbeta2 or Rem (Fig. 1). This increase was independent of sex; it was not different in male compared with female mice.

Measurement of protein and ionic current is necessary to evaluate functionally active channel proteins. To allow sufficient time for protein expression, cells were isolated for Western blot analysis and Ca2+ current measurement at 2 days after Ver pump implantation (2d-Ver). Western blot demonstrated a sixfold increase in CaV1.2 protein (Fig. 2).


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 2. Top: representative Western blot of CaV1.2 and GAPDH in vehicle- and Ver-treated animals. Bottom: densitometry of CaV1.2 normalized to GAPDH in vehicle (n = 4)- and Ver (n = 5)-treated animals. *P < 0.05.

 
Functionally active channel protein was assessed with current-voltage (I-V) relationships for ICa,L obtained by applying a series of 250-ms test pulses ranging from –40 to +50 mV in 5-mV increments from a holding potential of –40 mV at 0.2-Hz frequency. To isolate cells, hearts are removed and cells are rinsed free of acute Ver exposure for at least 1 h and up to 6 h. Thus Ver refers to the pretreatment. Consistent with the prediction from mRNA and protein changes, basal ICa,L from 2d-Ver mice was increased 1.4-fold relative to control mice (Fig. 3A). The increase of current occurred without a shift of voltage dependence.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 3. Calcium current (ICa)-voltage (V) relationship for control and chronic Ver-treated cardiac myocytes. A: top: representative original traces from control and chronic Ver-treated (thick traces) mice under basal conditions and after beta-acute beta-adrenergic stimulation (acute-beta-stim) (thin traces) with 1 µM isoproterenol (Iso). Bottom: average ICa-V relationships for control and chronic Ver treatment and after acute-beta-stim with 1 µM Iso. *Significant difference in current for basal states between the Ver group and the control group. B: maximal conductance (Gmax) in control and Ver-treated animals and after acute-beta-stim. Gmax was obtained from fitting the I-V data as in A (bottom) to a modified Boltzmann distribution of the form I(V) = Gmax*(V – Erev)/[1 + exp(V1/2 – V)/k], where Erev is reversal potential, V1/2 is activation midpoint potential, and k is the slope factor. C: membrane capacitance in control and after chronic Ver treatment for 2 days. Control measurements: 5 animals, 15 cells; Ver measurements: 4 animals, 18 cells. *P < 0.05.

 
A common mechanism for an increase of ICa,L is acute beta-adrenergic stimulation (Fig. 3A). Therefore we tested the response of ICa,L to acute Iso perfusion (to avoid a potentially confusing nomenclature, we refer to the use of acute 1 µM Iso perfusion of dispersed cells as acute-beta-stim). Acute-beta-stim is the immediate response of isolated cells to exposure of Iso with a duration of seconds. Cells from chronic Ver-treated animals responded to acute-beta-stim with a slight increase of maximal conductance (Gmax) and slight negative shift of I-V relationship (Fig. 3, A and B). The mean half-activation potentials (V1/2) for basal conditions were not statistically significant and were –10.1 ± 0.5 and –10.3 ± 1.0 mV for control and Ver mouse groups, respectively.

Chronic beta-adrenergic stimulation. Chronic Ver treatment increased HR and systolic BP (Table 1). The increase of HR is consistent with a compensatory baroreflex response that includes a compensatory increase of sympathetic stimulation. Therefore, we explored the effect of chronic beta-adrenergic stimulation by treatment with chronic Iso alone. Long-term Iso models are well established, although many previous studies used Iso treatment longer than 24 h (3, 12, 23, 30). After 24 h of Iso infusion heart/body weight increased, no change was observed in systolic and diastolic BP, but HR increased compared with both vehicle and background (Table 1). Cav1.2 mRNA levels were constant, whereas Rem levels increased twofold in hearts from 1d-Iso mice (Fig. 4).


Figure 4
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 4. Cardiac L-type and auxiliary subunit remodeling evaluated by real-time RT-PCR quantification in mice treated chronically with Iso for 1 day. Values are expressed as the relative quantification of the gene of interest vs. the internal control (cyclophilin A) and are represented as means ± SE. The numbers of mice used for experiments are shown within bars. *P < 0.05.

 
In Fig. 5A the peak values of ICa,L are plotted against their respective test potentials for basal conditions compared with acute-beta-stim, and the data are fitted to a modified Boltzmann distribution. The response of ICa,L to acute-beta-stim is eliminated by 2-day in vivo pretreatment. The maximal macroscopic conductance, manifested as the slope of the ascending limb of the I(V) curve, is significantly increased by acute-beta-stim in cells obtained from control animals, but it is unchanged in cells from Iso pump mice. Similarly, acute-beta-stim shifts the I(V) curve ~5 mV for control only. Figure 5B summarizes these effects, showing that Gmax of control cells was significantly increased during acute-beta-stim treatment (from 189 ± 10 to 257 ± 23 pS/pF; P < 0.005, n = 15) while acute-beta-stim had no effect on the Gmax of cells obtained from 2d-Iso. The mean V1/2 obtained was more negative in the presence of 1 µM Iso, but the differences were not significant. This loss of acute-beta-stim responsiveness confirms previous observations of the response of ICa,L to hypertrophic stimuli. As with 2d-Ver, Iso alone had no effect on ICa,L kinetics.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 5. ICa-V relationship for 2-day Iso-treated cardiac myocytes. A: top: representative original traces from control and Iso (2 day)-treated mice. Bottom: average ICa-V relationships for control and Iso (2 day) and after acute-beta-stim with 1 µM Iso. B: Gmax in control and Iso (2 day) and after acute-beta-stim with 1 µM Iso. C: membrane capacitance in control and after chronic Iso (2 day) treatment. *P < 0.05.

 
To further examine the homeostatic regulation of the Ca2+ channel we implanted pumps containing Ver and Iso in a single pump. Either Ver or Iso mice showed elevated HR, but Ver resulted in elevated BP. To counter this BP elevation we simultaneously infused mice with a combination of Iso + Ver. This combination resulted in a decrease of both systolic and diastolic BP along with an increase in heart rate (Table 1). The Iso + Ver treatment did not result in an increase in CaV1.2 mRNA expression such as that observed in the Ver mice (Fig. 6A). No significant changes were observed in basal ICa,L (Fig. 6B) or single-cell capacitance (Fig. 6D) compared with control. In addition, acute-beta-stim responsiveness was absent in these animals (Fig. 6C). The mean V1/2 values for basal conditions were not statistically significant and were –10.1 ± 0.5 and –10.4 ± 0.2 for the Control and Iso + Ver mouse groups, respectively. Thus changes in CaV1.2 RNA expression consistently reflect ICa,L changes. The extrinsic chronic beta-adrenergic stimulation provided by Iso apparently counteracted increases in CaV1.2 and the Ver-induced elevation of ICa,L.


Figure 6
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 6. A: cardiac L-type and auxiliary subunit remodeling evaluated by real-time RT-PCR quantification in mice treated chronically with Iso + Ver for 1 day. Values are expressed as the relative quantification of the gene of interest vs. the internal control (cyclophilin A) and are represented as means ± SE. The numbers of mice used for experiments are shown within bars. B: ICa-V relationship for control and chronic Iso + Ver-treated cardiac myocytes. Representative original traces from control and chronic Iso + Ver-treated (thick traces) mice under basal conditions and after acute-beta-stim (thin traces) with 1 µM Iso are shown. C: Gmax in control and Iso + Ver-treated animals and after acute-beta-stim. D: membrane capacitance in control and after chronic Iso + Ver treatment for 2 days. Iso + Ver pump: 4 animals, 13 cells. *P < 0.05.

 
ECG measurement. Ver increases CaV1.2 mRNA and protein and ICa,L and blunts acute-beta-stim responsiveness of ICa,L in isolated cells. To noninvasively investigate the effect of Ver on cardiac electrical activity, we evaluated the ECG from animals infused for 48 h with Ver, followed by a 24-h clearance period to reveal the functional consequences of CaV1.2 upregulation. ECG parameters in unrestrained Ver control mice are shown in Table 2. No significant differences were observed between Ver and control mice (Fig. 7, A and B). To mimic in vitro acute-beta-stim, we administered acute Iso via intraperitoneal injection. Control and Ver animals responded to acute-beta-stim with a prolonged R-R interval and an increase of amplitude of the P wave (Fig. 7, C and D). Although acute-beta-stim slowing of HR is unusual for large mammals, similar acute responses to intraperitoneal Iso in control mice were observed with ECG in previous studies (Ref. 9; Table 2). Despite the finding that the mean PR, QT, and QTc intervals were not significantly changed, consideration of the acute-beta-stim effect on an animal-by-animal basis (Fig. 7E) revealed an interesting effect that tends to correlate with the in vitro ICa,L recordings. Acute-beta-stim prolonged QTC in 80% (16/20) of control versus 41% (10/24) of Ver trials (P = 0.14, Fisher's exact test).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of this study is that long-term blockade of ICa,L by Ver increases ICa,L in cardiac myocytes isolated from mature hearts. This is in contrast to the well-established finding that chronic application of Iso in vivo has no effect on basal ICa,L but eliminates the acute beta-adrenergic receptor-induced increase of ICa,L. Ver treatment increases CaV1.2 mRNA and protein. In contrast, Iso has no effect on CaV1.2 mRNA but increases Rem (a negative regulator of Ca2+ channel expression). Moreover, Iso in combination with Ver prevents the Ver-induced mRNA and ICa,L increases. These data are consistent with the hypothesis that ventricular cardiac myocytes homeostatically regulate ICa,L. Our data suggest that chronic CCB elicits a compensatory upregulation of mRNA and protein that coincides with an increased ICa,L.

Persistent Ca2+ channel blockade. To examine homeostatic regulation of the Ca2+ channel in mouse ventricular myocardium, we implanted pumps containing Ver, which increased systolic BP and HR without affecting diastolic BP. Conversely, Ver is frequently used clinically to manage BP as a potent vasodilator. HR or pulse effects may be dependent on the mode of delivery and the pharmacokinetics of the Ca2+ channel antagonist. Ver has been shown to have minimal effect on HR when delivered via intracoronary injection. However, intravenous infusion of Ver results in a reflex increase in HR (43). To maintain cardiac output, HR would increase. In addition, vascular beds may constrict, increasing resistance in order to increase venous return. This increase in resistance would have a greater effect on systolic BP than diastolic BP (33). Systolic function has been shown to be effected by three principal mechanisms: Frank-Starling (force-length), force-frequency relations, and adrenergic stimulation. All three play a role in humans and larger mammals when enhanced cardiac output is needed. However, current research suggests that force-frequency relations and adrenergic stimulation play only a minor role in this response in mice. These hemodynamic differences highlight the need for further experimentation on larger mammals but should not detract from the result that cardiac ICa,L is homeostatically regulated.

Iso + Ver resulted in a decrease of both systolic and diastolic BP with an increase in HR. Ver and Iso function to dilate vessels in both arterial and venous circulation, decreasing total peripheral resistance and venous return, resulting in a decrease in BP. As a compensatory response, HR increases. Alternatively, the HR increase could be the result of the direct effect of Iso on the heart. Despite mouse-human differences in hemodynamic function, similar trends were observed with Ver treatment in patients scheduled for coronary angiography (37). Heart-to-body weight ratios were unchanged in Iso + Ver and decreased in Ver-only pumps. Ver has been shown to prevent increases in heart-to-body weight ratio observed in hypertrophy models (34, 42), and a decreased ventricular wet weight was demonstrated in chicken embryos treated with Ver (10). Despite similar, albeit unexpected, increases in HR by either Ver or Iso alone, Iso + Ver antagonized the Ver-induced increase of Ca2+ channels.

Surprisingly, mRNA and protein levels of CaV1.2 (the main pore-forming subunit of the Ca2+ channel) increased in Ver-treated mice (chronic Ca2+ channel blockade). We also observed an increase in ICa,L in Ver-treated mice. To our knowledge this is the first time an increase in Ca2+ current expression has been reported in response to chronic Ca2+ channel blockade. Iso + Ver treatment prevented the mRNA increase of the GTPase Rem present in the Iso-treated animals, suggesting that the upregulation of Rem may be mediated through changes in ICa,L. CaV1.2 and CaVbeta2 expression levels of the Iso + Ver-treated animals were similar to those observed with Iso alone.

The response of ICa,L to acute-beta-stim was diminished in Iso + Ver mice similar to the response observed in Iso mice. In Ver mice acute-beta-stim also resulted in a diminished responsiveness. Chronic treatment with beta-adrenergic agonists results in loss of receptors from the cell surface and degradation of receptor protein leading to decreased receptor expression (40).

beta-Adrenergic stimulation. In cardiac muscle, beta-adrenergic stimulation increases HR, force of contraction, and relaxation rate. Chronic beta-adrenergic stimulation, however, is commonly associated with hypertrophying heart in conditions such as heart failure. Our use of whole cell capacitance and acute Iso responsiveness measurements of cells dispersed from mice treated with Iso in vivo for periods as brief as 24–48 h reflects a hypertrophic phenotype at the cellular level. We also observed an increase in heart/body weight and HR at 24 h. Fan et al. (16) examined the transcriptional regulation of L-type Ca2+ channels in isolated rat myocytes. These in vitro studies observed coordinate regulation of CaV1.2, CaVbeta2, and {alpha}2{delta}. In vivo studies using whole animals are at odds with these in vitro findings, showing no change in CaV1.2 expression levels with treatment with Iso for up to 7 days (23); similarly, our results showed no change in CaV1.2 expression levels or Ca2+ current after 24-h Iso treatment. Aside from increasing ICa,L, CaVbetas play an important role in determining the behavior of L-type Ca2+ channels. They function to increase surface expression of the CaV1.2 ({alpha}-subunit) (8, 48) and produce hyperpolarizing shifts in the voltage dependence of channel activation.(5, 44) Rem has been shown to be a potent negative regulator of Ca2+ channel function through beta-subunit interaction (18). We speculate that the change observed in auxiliary subunits at 24 h may function to maintain a constant Ca2+ current.

Chronic beta-adrenergic stimulation eliminates the response of ICa,L to acute Iso. Ver also eliminated ICa,L responsiveness to acute-beta-stim, but the key difference was that Ver induced an increase of basal current, an increase of protein, and an increase of CaV1.2 mRNA that was not observed with Iso. It is possible that Ver activated a reflex response that increased sympathetic stimulation. However, Ver, in contrast to Iso, had selective effects on Ca2+ channel regulation.

ECG measurements. To evaluate the physiological implications of CCB we characterized ECG in control versus Ver and tested acute-beta-stim to parallel our in vitro studies. Although a selective increase of ICa,L would be expected to prolong QT interval (27), we measured no significant change of QT or QTc. It must be emphasized that action potential repolarization, which underlies QT interval, is the result of a balance of ICa,L and K+ currents. This balance is likely more delicate in humans and large mammals with longer plateau potentials. In the mouse transient outward K+ currents (Ito) such as that carried by KV4.2 dominate repolarization, and knockout of KV4.2 markedly prolongs QT interval (24). It is plausible that the relatively large Ito precludes observation of a 20% increase of ICa,L in the mouse. Also, we cannot rule out the possibility that CCB triggered multiple compensatory responses, and this is a focus of ongoing studies. Finally, it should be remembered that an accurate measure of complete repolarization cannot be obtained from a mouse ECG (14); thus ECG data showing no apparent effect of the mean QT intervals should be cautiously interpreted.

Limitations. ICa,L cannot be measured from cells in the intact heart. As a result, cells are removed from residual Ver blockade and channel reexpression may occur during isolation. For this scenario to obscure data interpretation requires that mechanisms of channel reexpression are different after CCB compared with control.

Studying ICa,L responses to drugs administered to intact animals is vastly more complex than cell culture models. Heart cells can be isolated and cultured for durations similar to those in our studies. Studying in vitro cultured cells alleviates numerous confounding variables presented by hemodynamic regulation in an intact animal. On the other hand, cardiac myocytes in primary culture can dedifferentiate (17). Thus it is impossible to know whether myocytes maintained in vitro reflect in vivo responses (16, 23).

We selected only two Ca2+ channel regulatory proteins for study in addition to the main pore-forming subunit. This sharply contrasts with genomic approaches that evaluate microarrays of cDNA for thousands of transcripts. Genomic approaches are a powerful tool to evaluate clusters of changes in response to stressors. However, omitting a single regulatory protein can skew microarray data interpretation. For example, while it is widely recognized that CaVbeta2 increases ICa,L, it is less well known that RGK proteins provide a robust block of ICa,L expression (2, 18). Thus it is imperative to determine ionic current levels (20). Importantly, our study reinforces the principle that multiple proteins sum to set a level of Ca2+ channel complex expression. Furthermore, assessment of mRNA levels as a readout of Ca2+ channel function will always be limited by our inability to identify all proteins in the Ca2+ channel complex (15). Moreover, posttranslational modifications of Ca2+ channels can dramatically alter ICa,L levels.

Summary. The main results of this study are as follows. 1) Chronic Ca2+ channel blockade significantly causes upregulation of CaV1.2 mRNA (the main pore-forming subunit) and results in an increase of Ca2+ current. 2) Chronic in vivo beta-adrenergic stimulation increases a negative (Rem) regulator of ICa,L without changing ICa,L levels. 3) Chronic Ca2+ channel block by Ver antagonizes the Iso-induced upregulation of Rem. Hemodynamic differences between mice and humans make it difficult to extrapolate the data to clinical situations. However, the first point has rather important implications considering the widespread clinical use of CCBs. Our results suggest that cardiac myocytes from the ventricular chamber of the heart respond to long-term Ca2+ channel block by increasing Ca2+ channel current. These data may provide insight into mechanisms for arrhythmia and sudden death in patients with sporadic noncompliance of a CCB drug regiment. Future experiments are needed on larger mammals examining the potential for action potential dispersion as a result of transmural or regional differences in ICa,L.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by National Heart, Lung, and Blood Institute Grants HL-074091 and HL-072936 (J. Satin).


   ACKNOWLEDGMENTS
 
We thank D. Rateri and D. Howatt from Dr. A. Daugherty's lab for technical assistance with blood pressure measurement and mini osmotic pump implantation. We also are thankful to K. Edenfield for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Schroder, Univ. of Kentucky, Dept. of Physiology, 800 Rose St., MS508, Lexington, KY 40536-0298 (e-mail: eschr0{at}uky.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Antzelevich C, Fish J. Electrical heterogeneity within the ventricular wall. Basic Res Cardiol 96: 517–527, 2001.[CrossRef][ISI][Medline]
  2. Beguin P, Nagashima K, Gonoi T, Shibasaki T, Takahashi K, Kashima Y, Ozaki N, Geering K, Iwanaga T, Seino S. Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature 411: 701–706, 2001.[CrossRef][Medline]
  3. Boknik P, Fockenbrock M, Herzig S, Knapp J, Linck B, Luss H, Muller FU, Muller T, Schmitz W, Schroder F, Neumann J. Protein phosphatase activity is increased in a rat model of long-term beta-adrenergic stimulation. Naunyn Schmiedebergs Arch Pharmacol 362: 222–231, 2000.[CrossRef][ISI][Medline]
  4. Brice N, Berrow N, Campbell V, Page K, Brickley K, Tedder I, Dolphin A. Importance of the different beta subunits in the membrane expression of the alpha1A and alpha2 calcium channel subunits: studies using a depolarization-sensitive alpha1A and alpha2 calcium channel. Eur J Neurosci 9: 749–759, 1997.[CrossRef][ISI][Medline]
  5. Castellano A, Wei X, Birnbaumer L, Perez-Reyes E. Cloning and expression of a neuronal calcium channel beta subunit. J Biol Chem 268: 12359–12366, 1993.[Abstract/Free Full Text]
  6. Catterall W. Structure and regulation of voltage-gated Ca2+ channel diversity. Annu Rev Neurosci 16: 521–555, 2000.
  7. Cheng HJ, Zhang ZS, Onishi K, Ukai T, Sane DC, Cheng CP. Upregulation of functional beta3-adrenergic receptor in the failing canine myocardium. Circ Res 89: 599–606, 2001.[Abstract/Free Full Text]
  8. Chien A, Zhao X, Shirokov R, Puri T, Chang C, Sun D, Rios E, Hosey M. Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels. J Biol Chem 270: 30036–30044, 1995.[Abstract/Free Full Text]
  9. Chu V, Otero JM, Lopez O, Morgan JP, Amende I, Hampton TG. Method for non-invasively recording electrocardiograms in conscious mice. BMC Physiol 1: 6, 2001.[CrossRef][Medline]
  10. Clark EB, Hu N, Turner DR, Litter JE, Hansen J. Effect of chronic verapamil treatment on ventricular function and growth in chick embryos. Am J Physiol Heart Circ Physiol 261: H166–H171, 1991.[Abstract/Free Full Text]
  11. Colecraft HM, Alseikhan B, Takahashi SX, Chaudhuri D, Mittman S, Yegnasubramanian V, Alvania RS, Johns DC, Marban E, Yue DT. Novel functional properties of Ca2+ channel beta subunits revealed by their expression in adult rat heart cells. J Physiol 541: 435–452, 2002.[Abstract/Free Full Text]
  12. Colucci W, Sawyer D, Singh K, Communal C. Adrenergic overload and apoptosis in heart failure: implications for therapy. J Card Fail 6: 1–7, 2000.[ISI][Medline]
  13. Crump SM, Correll RN, Schroder EA, Lester WC, Finlin BS, Andres DA, Satin J. The L-type calcium channel {alpha}-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current. Am J Physiol Heart Circ Physiol 291: H1959–H1971, 2006.[Abstract/Free Full Text]
  14. Danik S, Cabo C, Chiello C, Kang S, Wit AL, Coromilas J. Correlation of repolarization of ventricular monophasic action potential with ECG in the murine heart. Am J Physiol Heart Circ Physiol 283: H372–H381, 2002.[Abstract/Free Full Text]
  15. Demolombe S, Marionneau C, Le Bouter S, Charpentier F, Escande D. Functional genomics of cardiac ion channel genes. Cardiovasc Res 67: 438–447, 2005.[Abstract/Free Full Text]
  16. Fan IQ, Chen B, Marsh JD. Transcriptional regulation of L-type calcium channel expression in cardiac myocytes. J Mol Cell Cardiol 32: 1841–1849, 2000.[CrossRef][ISI][Medline]
  17. Fares N, Gomez JP, Potreau D. T-type calcium current is expressed in dedifferentiated adult rat ventricular cells in primary culture. CR Acad Sci III 319: 569–576, 1996.
  18. Finlin B, Andres D. Rem is a new member of the Rad- and Gem/Kir Ras-related GTP-binding protein family repressed by lipopolysaccharide stimulation. J Biol Chem 272: 21982–21988, 1997.[Abstract/Free Full Text]
  19. Finlin BS, Crump SM, Satin J, Andres DA. Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases. Proc Natl Acad Sci USA 100: 14469–14474, 2003.[Abstract/Free Full Text]
  20. Gaborit N, Steenman M, Lamirault G, Le Meur N, Le Bouter S, Lande G, Leger J, Charpentier F, Christ T, Dobrev D, Escande D, Nattel S, Demolombe S. Human atrial ion channel and transporter subunit gene expression remodeling associated with valvular heart disease and atrial fibrillation. Circulation 112: 471–481, 2005.[Abstract/Free Full Text]
  21. Gao T, Chien A, Hosey M. Complexes of the alpha 1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels. J Biol Chem 274: 2137–2144, 1999.[Abstract/Free Full Text]
  22. Girvin BG, Johnston GD. Comparison of the effects of a 7-day period of non-compliance on blood pressure control using three different antihypertensive agents. J Hypertens 22: 1409–1414, 2004.[CrossRef][ISI][Medline]
  23. Golden K, Ren J, Dean A, Marsh JD. Norepinephrine regulates the in vivo expression of the L-type calcium channel. Mol Cell Biochem 236: 107–114, 2002.[CrossRef][ISI][Medline]
  24. Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of Ito,f and Ito,s: early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 {alpha} subunit. Circ Res 87: 73–79, 2000.[Abstract/Free Full Text]
  25. Gurnett C, De Waard M, Campbell K. Dual function of the voltage-dependent Ca2+ channel alpha 2 delta subunit in current stimulation and subunit interaction. Neuron 16: 431–440, 1996.[CrossRef][ISI][Medline]
  26. Hofmann F, Biel M, Flockerzi V. Molecular basis for Ca2+ channel diversity. Annu Rev Neurosci 17: 399–418, 1994.[CrossRef][ISI][Medline]
  27. Johnson BD, Zheng W, Korach KS, Scheuer T, Catterall WA, Rubanyi GM. Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice. J Gen Physiol 110: 135–140, 1997.[Abstract/Free Full Text]
  28. Jones L, Wei S, Yue D. Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels. J Gen Physiol 112: 125–143, 1998.[Abstract/Free Full Text]
  29. Lacerda A, Kim H, Ruth P, Perez-Reyes E, Flockerzi V, Hofmann F, Birnbaumer L, Brown A. Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature 352: 527–530, 1991.[CrossRef][Medline]
  30. Leone M, Albanese J, Martin C. Positive inotropic stimulation. Curr Opin Crit Care 8: 395–403, 2002.[CrossRef][Medline]
  31. Letts V, Felix R, Biddlecome G, Arikkath J, Mahaffey C, Valenzuela A, Bartlett F 2nd, Mori Y, Campbell K, Frankel W. The mouse stargazer gene encodes a neuronal Ca2+ channel gamma subunit. Nat Genet 19: 340–347, 1998.[CrossRef][ISI][Medline]
  32. Lipsitz LA, Gagnon M, Vyas M, Iloputaife I, Kiely DK, Sorond F, Serrador J, Cheng DM, Babikian V, Cupples LA. Antihypertensive therapy increases cerebral blood flow and carotid distensibility in hypertensive elderly subjects. Hypertension 45: 216–221, 2005.[Abstract/Free Full Text]
  33. London GM, Guerin AP. Influence of arterial pulse and reflected waves on blood pressure and cardiac function. Am Heart J 138: 220–224, 1999.[CrossRef][Medline]
  34. Mandarim-de-Lacerda CA, Meirelles Pereira LM. Volume-weighted mean nuclear volume and numerical nuclear density in the cardiomyocyte following enalapril and verapamil treatment. Virchows Arch 438: 92–95, 2001.[CrossRef][ISI][Medline]
  35. Mitchell G, Andreas J, Koren G. Measurement of heart rate and A-T interval in the conscious mouse. Am J Physiol Heart Circ Physiol 274: H747–H751, 1998.[Abstract/Free Full Text]
  36. Olcese R, Qin N, Schneider T, Neely A, Wei X, Stefani E, Birnbaumer L. The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron 13: 1433–1438, 1994.[CrossRef][ISI][Medline]
  37. Oldenburg O, Eggebrecht H, Herrmann J, Naber CK, Haude M, Erbel R, Baumgart D. Dose-dependent effects of intracoronary verapamil on systemic and coronary hemodynamics. Cardiovasc Drugs Therapy 14: 651–655, 2000.[CrossRef]
  38. Pavlovic K, Benc D, Kmezic-Grujin J, Stojiljkovic J, Cemerlic-Adic N, Miljkovic T, Mesaros P. Fosinopril and amlodipine in the treatment of isolated systolic hypertension. Med Pregl 57: 45–53, 2004.[CrossRef][Medline]
  39. Perez-Reyes E, Castellano A, Kim H, Bertrand P, Baggstrom E, Lacerda A, Wei X, Birnbaumer L. Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel. J Biol Chem 267: 1792–1797, 1992.[Abstract/Free Full Text]
  40. Post SR, Hammond HK, Insel PA. Adrenergic receptors and receptor signaling in heart failure. Annu Rev Pharmacol Toxicol 39: 343–360, 1999.[CrossRef][ISI][Medline]
  41. Richard S, Leclercq F, Lemaire S, Piot C, Nargeot J. Ca2+ current in compensated hypertrophy and heart failure. Cardiovasc Res 37: 300–311, 1998.[Abstract/Free Full Text]
  42. Sah R, Oudit GY, Nguyen TTT, Lim HW, Wickenden AD, Wilson GJ, Molkentin JD, Backx PH. Inhibition of calcineurin and sarcolemmal Ca2+ influx protects cardiac morphology and ventricular function in Kv4.2N transgenic mice. Circulation 105: 1850–1856, 2002.[Abstract/Free Full Text]
  43. Schmieder R, Messerli F, Garavaglia G, Nunez B. Cardiovascular effects of verapamil in patients with essential hypertension. Circulation 75: 1030–1036, 1987.[Abstract/Free Full Text]
  44. Singer D, Biel M, Lotan I, Flockerzi V, Hofmann F, Dascal N. The roles of the subunits in the function of the calcium channel. Science 253: 1553–1557, 1991.[Abstract/Free Full Text]
  45. Wei SK, Colecraft H, DeMaria C, Peterson B, Zhang R, Kohout T, Rogers T, Yue D. Ca2+ channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells. Circ Res 86: 175–184, 2000.[Abstract/Free Full Text]
  46. Wei SK, Ruknudin A, Hanlon SU, McCurley JM, Schulze DH, Haigney MC. Protein kinase A hyperphosphorylation increases basal current but decreases beta-adrenergic responsiveness of the sarcolemmal Na+-Ca2+ exchanger in failing pig myocytes. Circ Res 92: 897–903, 2003.[Abstract/Free Full Text]
  47. Wickenden AD, Kaprielian R, Kassiri Z, Tsoporis JN, Tsushima R, Fishman GI, Backx PH. The role of action potential prolongation and altered intracellular calcium handling in the pathogenesis of heart failure. Cardiovasc Res 37: 312–323, 1997.[CrossRef][ISI]
  48. Yamaguchi H, Hara M, Strobeck M, Fukasawa K, Schwartz A, Varadi G. Multiple modulation pathways of calcium channel activity by a beta subunit. Direct evidence of beta subunit participation in membrane trafficking of the alpha1C subunit. J Biol Chem 273: 19348–19356, 1998.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/H1906    most recent
00793.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schroder, E.
Right arrow Articles by Satin, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schroder, E.
Right arrow Articles by Satin, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.